Many bacteriophage and integrative plasmids encode site-specific recombination systems that enable the stable incorporation of their genome into those of their hosts and excision of the genome from the host genome. In these systems, the minimal requirements for the recombination reaction are a recombinase enzyme, which catalyzes the recombination event, and two recombination sites (Sadowski (1986) J. Bacteriol. 165: 341-347; Sadowski (1993) FASEB J. 7: 760-767). For phage integration systems, these are referred to as attachment (alt) sites, with an attP element from phage DNA and the attB element present in the bacterial genome. The two attachment sites can share as little sequence identity as a few base pairs. The recombinase protein binds to both att sites and catalyzes a conservative and reciprocal exchange of DNA strands that result in integration of the circular phage or plasmid DNA into host DNA. Additional phage or host factors, such as the DNA bending protein IHF, integration host factor, may be required for an efficient reaction (Friedman (1988) Cell 55:545-554; Finkel & Johnson (1992) Mol. Microbiol. 6: 3257-3265). Phage integrases, in association with other host and/or phage factors, also excise the phage genome from the bacterial genome during the lytic phase of bacteriophages growth cycle. Several methods have been developed allowing the manipulation of mammalian genomes in order to elucidate the relevance and function of particular genes of interest. Among them, the development of transgenic mouse strains and gene-targeting technologies have turned out to be particularly useful (Brandon, E. P., Idzerda, R. L. and McKnight, G. S. (1995) Curr Biol, 5, 625-34; Brandon, E. P., Idzerda, R. L. and McKnight, G. S. (1995) Curr Biol, 5, 758-65). These techniques have undergone a new advance with the characterization and application of site-specific recombinases (Kilby, N. J., Snaith, M. R. and Murray, J. A. (1993) Trends Genet, 9, 413-21).
Site-specific recombinases can be separated into two major families. The first one (the Int family or tyrosine recombinase family) comprises those enzymes that catalyze recombination between sites located either in the same DNA molecule (intramolecular recombination leading to resolution, excision, or inversion) or in separate DNA molecules (intermolecular recombination leading to integration) (Sauer, B. (1993) Methods Enzymol, 225, 890-900; Dymecki, S. M. (1996) Proc Natl Acad Sci U(SA, 93, 6191-6; Abremski, K. and Hoess, R. (1984) J Biol Chem, 259, 1509-14; Nash, H. A. (1996) in Escherichia coli and Salmonella cellular and molecular biology, ed. F. C. Neidhart, R. I. Curtis, J. L. Ingraham, E. C. C. Lin, K. B. Low, B. Magasanik, W. S. Rezaikoff, M. Riley, M. Schaechter and H. E. Umbager (A.S.M. Press, Washington D.C.), pp. 2363-7). The latter property has been exploited to allow targeted insertion of specific sequences in precise locations (Sauer, B. and Henderson, N. (1990) The New Biologist, 2, 441-9; Fukushige, S. and Sauer, B. (1992) Proc. Natl. Acad. Sci. USA, 89, 7905-9). The recombinases that have been used for manipulating mammalian genomes have been mainly the Cre and the Flp proteins, which belong to the Int family (Kilby, N. J., Snaith, M. R. and Murray, J. A. (1993) Trends Genet, 9, 413-21). The target sequences for these enzymes, named loxP sites for the Cre enzyme and FRT for the Flp enzyme, consist of a short inverted repeat to which the protein binds. The recombination process is operative through long distances (up to 70 kb) in the genome. Using these enzymes, several authors have reported site- and tissue-specific DNA recombination in murine models (DiSanto, J. P., Muller, W., Guy, G. D., Fischer, A. and Rajewsky, K. (1995) Proc Natl Acad Sci USA, 92, 377-81; Gu, H., Marth, J. D., Orban, P. C., Massmann, H. and Rajewsky, K. (1994) Science, 265, 103-6; Kuhn, R., Schwenk, F., Aguet, M. and Rajewsky, K. (1995) Science, 269, 1427-9; Orban, P. C., Chui, D. and Marth, J. D. (1992) Proc. Natl. Acad. Sci. USA, 89, 6861-5), chromosomal translocations in plants and animals (Deursen, J. v., Fornerod, M., Rees, B. v. and Grosveld, G. (1995) Proc. Natl. Acad. Sci. USA, 92, 7376-80; Medberry, S. L., Dale, E., Qin, M. and Ow, D. W. (1995) Nucleic Acids Res, 23, 485-90; Osborne, B. I., Wirtz, U. and Baker, B. (1995) Plant J, 7, 687-701) and targeted induction of specific genes (Pichel, J. G., Lakso, M. and Westphal, H. (1993) Oncogene, 8, 3333-42). The Cre-loxP system has also been used in combination with inducible promoters, such as the interferon gamma inducible promoter, that was used to provoke gene ablation in liver with high efficiency and to a less extent in other tissues (Kuhn, R., Schwenk, F., Aguet, M. and Rajewsky, K. (1995) Science, 269, 1427-9). This site-specific recombination system, however, only allows the induction of a reduced number of recombination events in the same genome. Since each recombination reaction leaves a target sequence for the recombinase in the genome at the crossover site, and because recombinases (e.g. Cre and Flp) can catalyze intermolecular recombination, the whole process may lead to undesired chromosomal rearrangements.
The second family of recombinases are collectively termed resolvases/invertases family or serine family (Grindley, N. D. F. (1994) in Nucleic Acids and Molecular Biology, ed. F. Eckstein and D. M. J. Lilley (Springer-Verlag, Berlin), pp. 236-67, (Smith, M. C. and Thorpe, H. M. (2000) Mol. Microbiol., 44, 299-307)). These site-specific recombinases, which include enzymes that catalyze intramolecular and intermolecular reactions, could have an advantage over the Int family of recombinases. Serine recombinases that catalyze phage integration (integrases) are especially well adapted for use as genetic engineering tools. So far three serine recombinases, ϕC31, R4 and TP901-1, have been examined in mammalian cells (Groth, A. C. and Calos, M. P. (2004) J. Mol. Biol. 335, 667-678). These recombinases were observed to be autonomous, to have simple att sequences and have the ability to function in mammalian cells. As little or no recombination between any combination of sites other than attP or attB has been observed, the integrations are unidirectional and there is a high integration frequency. Serine recombinases provide a significant advantage over the prior recombination systems employing the use of members of the Int family of recombinases. These enzymes have numerous applications. One way is the placement of att sites into the genome of an organism and use as targets for recombination.
Applicant has identified novel serine recombinases that demonstrate robust activity in various mammalian cells and in plant cells, as well as the ability to stably integrate a polynucleotide into the genome of a host cell or excise a polynucleotide from the genome of a host cell.